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F Grinnell

Publications and source records attributed to F Grinnell.

At least 37 records · Page 2Linked to original sources
PubMed1996

Fibronectin degradation in chronic wounds depends on the relative levels of elastase, alpha1-proteinase inhibitor, and alpha2-macroglobulin.

The goal of our studies was to learn about the mechanism of fibronectin degradation in chronic ulcers. We found that the appearance of fibronectin fragments in chronic ulcer wound fluid correlated with elevated levels of elastase and cleavage of the proteinase inhibitors alpha2-macroglobulin (alpha2-M) and alpha 1-proteinase inhibitor (alpha1-P1). Some wound fluid samples retained the capacity to degrade fibronectin in vitro. Degradation of fibronectin by these samples was blocked by specific inhibitors of neutrophil elastase but not by inhibitors of metalloproteinases. Addition of human neutrophil elastase to mastectomy fluid, an acute wound fluid, resulted in formation of alpha1-PI and alpha2-M complexes and cleavage products resembling those observed in chronic wound fluid. Moreover, degradation of fibronectin and processing of matrix metalloproteinase MMP-9 occurred under these conditions. Taken together, our findings suggest that elevated levels of neutrophil elastase are responsible for fibronectin degradation in the chronic wound environment.

Adult↗
PubMed1995

Non-muscle myosin II heavy chain has a cryptic cell-adhesion domain.

We have discovered a cryptic cell-adhesion domain in non-muscle myosin II heavy chain. A 205 kDa cell-adhesion-promoting polypeptide (p205) was extracted from BHK cells by Nonidet P-40 or Dounce homogenization. Adhesion to p205 was specifically inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro, indicating a role for the Arg-Gly-Asp cell-adhesion motif. Purified p205 was identified as non-muscle myosin II heavy chain, based on sequence analysis and on the cross-reactivity of p205 with anti-(bovine trachea myosin) antibodies. Further experiments showed that the heavy chain of purified myosin II has cell-adhesion-promoting activity in a cell-blotting assay, and cross-reacted with anti-p205 antibodies. Finally, the adhesion domain was located in the tail portion of myosin II heavy chain, where an Arg-Gly-Asp-containing sequence can be found.

Amino Acid Sequence↗
PubMed1995

Fibronectin receptor internalization and AP-2 complex reorganization in potassium-depleted fibroblasts.

Potassium-depleted fibroblasts are unable to develop polarized morphology and lack coated pits. Experiments were carried out to measure internalization of fibronectin receptors (FNR) in potassium-depleted cells and possible association of FNR with AP-2 complexes after adding potassium back to the cells, which restores cell polarization. AP-2 complexes are the cell surface component of coated pits that contain both clathrin and membrane receptor binding domains. Potassium-depleted fibroblasts endocytosed antibody-tagged FNR and also internalized fluorescent fibronectin that previously had been adsorbed to the substratum. During cell polarization, antibody-tagged FNR reorganized into fibrillar structures along stress fibers beginning from nucleation sites at cell margins. Plasma membrane AP-2 complexes, which were undetectable in potassium-depleted cells, reappeared at the cell surface above the nucleus and then spread toward the cell margins. The results show that endocytosis of FNR can occur at least partially by a coated pit-independent mechanism.

Actins↗
PubMed1995

Treatment of human fibroblasts with vanadate and platelet-derived growth factor in the presence of serum inhibits collagen matrix contraction.

Human fibroblasts cultured in an anchored collagen matrix reorganize the matrix and develop stress. Upon experimental release of the matrix, cells contract the matrix, a phenomenon that has been studied as an in vitro model of wound contraction. We have found that treatment of fibroblasts in stressed collagen matrices with vanadate and platelet-derived growth factor (PDGF) in serum-containing medium inhibits the ability of cells to contract the matrix. Vanadate/PDGF/serum stimulation did not block contraction immediately, but rather resulted in generation of an inhibitory signal that developed over a period of 80 min. The signal was highly specific since other factors such as lysophosphatidic acid and epidermal growth factor were unable to replace PDGF or serum. The presence of vanadate also blocked dephosphorylation of p-Tyr-PDGF receptors after PDGF/serum stimulation and caused accumulation of tyrosine-phosphorylated proteins in the cells. In parallel experiments, fibroblasts in monolayer culture were found to undergo reorganization of their actin cytoskeleton when treated with vanadate in the presence of PDGF and serum-containing medium. Our results suggest that a p-Tyr signaling pathway is important in the regulation of wound contraction.

Actins↗
PubMed1995

Role of phospholipase D in the cAMP signal transduction pathway activated during fibroblast contraction of collagen matrices.

Fibroblast contraction of stressed collagen matrices results in activation of a cAMP signal transduction pathway. This pathway involves influx of extracellular Ca2+ ions and increased production of arachidonic acid. We report that within 5 min after initiating contraction, a burst of phosphatidic acid release was detected. Phospholipase D was implicated in production of phosphatidic acid based on observation of a transphosphatidylation reaction in the presence of ethanol that resulted in formation of phosphatidylethanol at the expense of phosphatidic acid. Activation of phospholipase D required extracellular Ca2+ ions and was regulated by protein kinase C. Ethanol treatment of cells also inhibited by 60-70% contraction-dependent release of arachidonic acid and cAMP but had no effect on increased cAMP synthesis after addition of exogenous arachidonic acid or on phospholipase A2 activity measured in cell extracts. Moreover, other treatments that inhibited the burst of phosphatidic acid release after contraction--chelating extracellular Ca2+ or down-regulating protein kinase C--also blocked contraction activated cyclic AMP signaling. These results were consistent with the idea that phosphatidic acid production occurred upstream of arachidonic acid in the contraction-activated cAMP signaling pathway.

Arachidonic Acid↗
PubMed1994

Stress relaxation of fibroblasts activates a cyclic AMP signaling pathway.

Mechanical force regulates gene expression and cell proliferation in a variety of cell types, but the mechanotransducers and signaling mechanisms involved are highly speculative. We studied the fibroblast signaling mechanism that is activated when cells are switched from mechanically stressed to mechanically relaxed conditions, i.e., stress relaxation. Within 10 min after initiation of stress relaxation, we observed a transient 10-20-fold increase in cytoplasmic cyclic AMP (cAMP) and a threefold increase in protein kinase A activity. The increase in cAMP depended on stimulation of adenylyl cyclase rather than inhibition of phosphodiesterase. Generation of cAMP was inhibited by indomethacin, and release of arachidonic acid was found to be an upstream step of the pathway. Activation of signaling also depended on influx of extracellular Ca2+ because addition of EGTA to the incubations at concentrations just sufficient to exceed Ca2+ in the medium inhibited the stress relaxation-dependent increase in free arachidonic acid and cAMP. This inhibition was overcome by adding CaCl2 to the medium. On the other hand, treating fibroblasts in mechanically stressed cultures with the calcium ionophore A23187-stimulated arachidonic acid and cAMP production even without stress relaxation. In summary, our results show that fibroblast stress relaxation results in activation of a Ca(2+)-dependent, adenylyl cyclase signaling pathway. Overall, the effect of stress relaxation on cAMP and PKA levels was equivalent to that observed after treatment of cells with forskolin.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Identification of neutrophil elastase as the proteinase in burn wound fluid responsible for degradation of fibronectin.

To identify proteinases responsible for fibronectin degradation in the wound environment we studied wound fluid obtained from burn patients. Immunoblotting experiments showed that extensive degradation of fibronectin had occurred in some burn wound fluid samples, in which case intact fibronectin molecules were undetectable, and the largest fibronectin fragment was 116 kDa. The 116-kDa fragment as well as a smaller 90-kDa fragment contained the fibronectin cell binding domain. These burn-fluid samples degraded freshly added fibronectin. Activity of the fibronectin-degrading enzyme was blocked by a broad-spectrum serine proteinase inhibitor or by specific neutrophil elastase inhibitors but not by metalloproteinase inhibitors or inhibitors of trypsin-like or chymotrypsin-like serine proteinases. Enzyme activity also was neutralized by antibodies against human neutrophil elastase. Incubation of fibronectin with burn wound fluid or purified human neutrophil elastase generated similar fibronectin-degradation products. Finally, direct assay of burn-wound-fluid samples with a synthetic elastase substrate showed a correlation between fluid-phase elastase activity and fibronectin degradation. Based on these findings, we conclude that burn-wound-fluid elastase is responsible for extensive fibronectin degradation. Acute elevation of elastase did not appear to hinder normal wound repair.

Burns↗
PubMed1994

Metalloproteinase activation cascade after burn injury: a longitudinal analysis of the human wound environment.

In this paper, we present a longitudinal study on metalloproteinases in wound-fluid samples collected from three patients with partial- to full-thickness burn wounds. Gelatin zymography showed that 92-kDa gelatinase (MMP-9) and its 225-kDa complex could be detected in burn fluid beginning as early as 4-8 h after injury. Marked increases in MMP-9 levels as well as activation of the proenzyme occurred between day 0 and day 2. The 72-kDa gelatinase (MMP-2) proenzyme was not detected until day 2 and activated enzyme did not appear until day 4. Stromelysin (MMP-3), both proenzyme and activated-enzyme forms, was first observed on day 4. Fluid-phase proteinase activity detected by azocoll degradation roughly corresponded with the level of stromelysin rather than the gelatinases. Our results provide evidence for a regulated metalloproteinase activation cascade following acute traumatic injury and demonstrate in vivo expression of metalloproteinase activity.

Adult↗
PubMed1993

Detection of the actin scavenger system in burn wound fluid.

The intravascular actin scavenger system depolymerizes and sequesters actin released after tissue injury. Studies were carried out to determine if this system is active in the extracellular space during wound repair. Using burn wound fluid as a noninvasive means for analyzing the wound environment, we measured actin accumulation and actin complex formation with the plasma proteins responsible for scavenger function. Actin at concentrations as high as 0.25 mg/ml ( approximately 5 micromol/L) was found in burn wound fluid samples from 9 of 11 patients. Wound fluid also contained the two plasma proteins that bind actin-gelsolin (both plasma and cytoplasmic forms) and Gc protein. Because actin in wound fluid was complexed with gelsolin and Gc protein, we conclude that the components of the actin scavenger system are functional in wound tissue. In addition, proteolysis of gelsolin, but not actin or Gc protein, appeared to occur at the wound site. Gelsolin proteolysis was accompanied by the appearance of 49 kd gelsolin fragments, and wound fluid samples lacking intact gelsolin also contained high metalloproteinase levels.

Journal Article↗
PubMed1993

Depletion of intracellular potassium disrupts coated pits and reversibly inhibits cell polarization during fibroblast spreading.

To learn more about the possible role of the coated pits endocytic pathway in cell adhesion, we studied attachment and spreading of fibroblasts whose coated pits were disrupted by depletion of intercellular potassium. Fibroblasts incubated in suspension in potassium-free medium lost 80% of their intracellular potassium within 10 min and showed disrupted coated pits based on fluorescence staining of clathrin. Potassium-depleted cells attached and spread on fibronectin-coated substrata over the same time course (15 min-2 h) as control cells. Unlike controls, however, potassium-depleted fibroblasts attained a radial morphology with circumferentially organized actin filament bundles and were unable to make the transition to a polarized morphology with stress fibers. In the radially spread fibroblasts, fibronectin receptors and vinculin colocalized in focal adhesion sites and appeared to be membrane insertion points for circumferentially arranged actin filament bundles, but these sites were much smaller than the focal adhesion plaques in polarized cells. The effects of potassium depletion on cell adhesion were reversible. Within 1 h after switching K(+)-depleted fibroblasts to medium containing KCl, cells developed a polarized morphology with actin stress fibers inserting into focal adhesion plaques. Coated pits also reformed on the cell surface during this time. Because formation of focal adhesion plaques preceded reappearance of clathrin-coated pits at the cell margins, it seems unlikely that coated pits play a direct role in adhesion plaque assembly. Polarization of fibroblasts upon addition of KCl was inhibited by ouabain showing that intracellular potassium was required for activity. Polarization also was inhibited when potassium-depleted cells were switched to potassium-containing medium under hypertonic or acidified conditions, both of which have been shown to inhibit receptor-mediated endocytosis. Our results suggest that the coated pit endocytic pathway is not required for initial attachment, spreading, and formation of focal adhesions by fibroblasts, but may play a role in cell polarization.

Actins↗
PubMed1993

Decreased level of PDGF-stimulated receptor autophosphorylation by fibroblasts in mechanically relaxed collagen matrices.

The goal of our studies was to characterize the interrelationship between extracellular matrix organization and fibroblast proliferation in response to growth factors. We compared fibroblasts in monolayer culture with cells in contracted collagen matrices that were mechanically stressed or relaxed. In response to platelet-derived growth factor (PDGF), DNA synthesis by fibroblasts in mechanically relaxed collagen matrices was 80-90% lower than in monolayer culture and 50% lower than in mechanically stressed matrices. Fibroblasts in monolayer and contracted collagen matrix cultures contained similar levels of PDGF receptors, but differed in their autophosphorylation response. Cells in mechanically relaxed matrices showed lowest levels of autophosphorylation, 90% less than cells in monolayer culture. Experiments comparing receptor expression and capacity for PDGF-stimulated autophosphorylation showed that cells in mechanically relaxed collagen matrices never developed normal receptor autophosphorylation. Furthermore, when mechanically stressed collagen matrices were switched to mechanically relaxed conditions, capacity for receptor autophosphorylation decreased within 1-2 h and remained low. Based on immunomicroscopic observations and studies on down-regulation of receptors by PDGF binding, it appeared that most PDGF receptors in monolayer or contracted collagen matrix cultures were localized on the cell surface and accessible to PDGF binding. In related studies, we found that EGF receptors of fibroblasts in mechanically relaxed collagen matrices also showed low levels of autophosphorylation in response to EGF treatment. Based on these results, we suggest that mechanical interactions between cells and their surrounding matrix provide regulatory signals that modulate autophosphorylation of growth factor receptors and cell proliferation.

Cell Membrane↗
PubMed1993

Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9.

The purpose of the present research was to determine if metalloproteinase levels were elevated in human chronic wound fluid. Samples of blood and wound fluid from acute (mastectomy) and chronic (leg ulcer) wounds were collected, and metalloproteinase profiles of the samples were determined by gelatin zymography. Compared to serum, acute wound fluid (mastectomy fluid) contained markedly increased levels (five-to tenfold) of several metalloproteinases including 72-kDa and 94-kDa gelatinases (MMP-2 and MMP-9). In chronic wound fluid, not only were these enzyme levels increased another five- to tenfold over mastectomy fluid, but also both activated enzyme and proenzyme species appeared to be present. Our results suggest that non-healing ulcers develop an environment containing high levels of activated metalloproteinases, which may result in chronic tissue turnover and failed wound closure.

Acute Disease↗
PubMed1993

Stress-relaxation of fibroblasts in collagen matrices triggers ectocytosis of plasma membrane vesicles containing actin, annexins II and VI, and beta 1 integrin receptors.

To learn about the effects of tension on fibroblast function, we have been studying initial cellular responses to stress-relaxation. Human foreskin fibroblasts were cultured in anchored collagen matrices for 2 days, during which time mechanical stress developed. Subsequently, the matrices were dislodged; thereby allowing stress to dissipate. Within 5 min after initiating stress-relaxation, fibroblasts retracted their pseudopodia. At this time, we observed the disappearance of cellular stress fibers and the formation of actin clusters along the cell margins. The actin was found to be located inside 200 nm diameter vesicles that were budding from the cell surface. Vesicles isolated from the matrix after stress-relaxation contained prominent 24 kDa, 36 kDa (doublet), 45 kDa, and 135 kDa polypeptides. The 45 kDa polypeptide was the major component in the Triton-insoluble vesicle fraction and appeared to be actin. The 36 kDa (doublet) polypeptide, which was found predominantly in the Triton-soluble vesicle fraction, was identified as annexin II. Vesicles also contained annexin VI and beta 1 integrin receptors but not tubulin, vimentin, vinculin or annexin I. The results suggest that stress-relaxation of fibroblasts induces a novel ectocytotic process involving transient budding of intact, plasma membrane vesicles from the cell cortex. On the basis of their morphological and biochemical features, these vesicles may be analogous to the 'matrix vesicles' released by chondrocytes and could play a role in extracellular matrix remodeling after wound contraction.

Actins↗
PubMed1992

Degradation of fibronectin and vitronectin in chronic wound fluid: analysis by cell blotting, immunoblotting, and cell adhesion assays.

We used a combination of cell blotting, immunoblotting, and cell adhesion assays to analyze fibronectin and vitronectin in wound fluid from acute and chronic wounds. Acute wound fluid (e.g., suction blister fluid, mastectomy fluid) contained intact fibronectin and vitronectin as major cell adhesion proteins. In marked contrast, chronic wound fluid samples from three of 11 patients with venous stasis ulcers showed complete degradation of vitronectin and degradation of fibronectin into small molecular mass polypeptides less than 125 kDa. Three of these polypeptides--54, 93, and 125 kDa--were biologically active in promoting cell attachment and were recognized by monoclonal antibodies that bind fibronectin near the arg-gly-asp (RGD) domain. In wound fluid samples from the other eight of 11 patients, only slight degradation of vitronectin and fibronectin occurred, which resulted in a mixture of mostly intact molecules along with large fragments. Intact fibronectin in chronic wound fluid samples contained the ED-A domain, which showed that fibronectin synthesis occurred locally in the wound bed. Wound fluid containing extensively degraded vitronectin and fibronectin reversibly inhibited cell adhesion, and excess fetal bovine serum, but not purified fibronectin, neutralized the inhibitory effect. We suggest that protease activity in some chronic wounds may cause degradation of adhesion proteins and prevent cell adhesion necessary for normal wound closure.

Animals↗
PubMed1992

Altered glycosylation and cell surface expression of beta 1 integrin receptors during keratinocyte activation.

We studied the mechanism by which cell adhesiveness becomes activated when keratinocytes are removed from skin and placed into cell culture. Our results suggest that activation involves altered beta 1 integrin subunit glycosylation accompanied by an increase in cell surface beta 1 integrin receptors. Activated keratinocytes contained two forms of the beta 1 integrin subunit, approximately 93 kDa and approximately 113 kDa. As shown by pulse-chase experiments, the smaller represented the cytoplasmic precursor of the larger, and only the 113 kDa mature form was detected in integrin receptors expressed at the cell surface. Pre-activated keratinocytes contained beta 1 integrin subunits ranging from approximately 97 to 110 kDa. These beta 1 subunits had been processed through the Golgi, based on resistance to endoglycosidase-H treatment, and were not converted to 113 kDa subunits during subsequent cell culture. Experiments with endoglycosidase-F showed that differences in the apparent sizes of beta 1 integrin subunits observed in pre-activated and activated keratinocytes could be attributed to differences in subunit glycosylation. Smaller beta 1 subunits found in pre-activated keratinocytes, like the precursor beta 1 subunits of activated cells, appeared to be less efficient in reaching the cell surface. Overall, a approximately 10-fold increase in the level of cell surface integrin receptors occurred concomitant with the increased proportion of 113 kDa beta 1 subunits found in activated cells. Endoglycosidase-F experiments also indicated that there were changes in keratinocyte alpha subunits associated with beta 1. In related experiments, keratinocytes cultured in low Ca2+, serum-free MCDB medium for 4 days proliferated but their adhesiveness did not become activated. Therefore, keratinocyte proliferation and activation of adhesion are regulated separately. Finally, substantial activation of keratinocytes was observed when serum was added to cells cultured in MCDB with serum, indicating a role for serum factors in the activation process.

Animals↗